Photonic crystals, also commonly referred to as photonic bandgap structures, are periodic dielectric structures exhibiting a spatially periodic variation in refractive index that forbids propagation of certain frequencies of incident electromagnetic radiation. The photonic band gap of a photonic crystal refers to the range of frequencies of electromagnetic radiation for which propagation through the structure is prevented in particular directions. A photonic crystal structure may be designed to exhibit extraordinarily high reflection efficiency at particular wavelengths, at which optical standing waves develop and resonate within the photonic crystal structure. Such optical resonances are known to occur at the wavelengths adjacent to the photonic band gap, sometimes referred to as the photonic band edge. The spatial arrangement and refractive indices of these structural domains generate photonic bands gaps that inhibit propagation of electromagnetic radiation centered about a particular frequency.
This anomalous resonant phenomenon (termed guided-mode resonance) arises due to the introduced periodicity which allows phase-matching of externally incident radiation into modes that can be reradiated into free-space. Due to the fact that these modes possess finite lifetimes within such structures, they are referred to as ‘leaky eigenmodes’ of the structures. More recently, guided-mode resonances have been studied in crossed gratings or two-dimensional (2D) photonic crystal (PC) slabs. The leaky nature of these modes has been exploited towards the development of light emitting diodes (LEDs) with improved extraction efficiency, biosensors (see Cunningham et al., Colorimetric Resonant Reflection as a Direct Biochemical Assay Technique, Sensors and Actuators B, 2002, 81, pgs 316-328 (2002)) and vertically emitting lasers.
The ability of photonic crystals to provide high quality factor (Q) resonant light coupling, high electromagnetic energy density, and tight optical confinement can also be exploited to produce highly sensitive biochemical sensors. Here, Q is a measure of the sharpness of the peak wavelength at the resonant frequency. Photonic crystal biosensors are designed to allow a liquid test sample to penetrate the periodic lattice, and to tune the resonant optical coupling condition through modification of the surface dielectric constant of the crystal through the attachment of biomolecules or cells. Due to the high Q of the resonance, and the strong interaction of coupled electromagnetic fields with surface-bound materials, several of the highest sensitivity biosensor devices reported are derived from photonic crystals. Such devices have demonstrated the capability for detecting molecules with molecular weights less than 200 Daltons (Da) with high signal to noise margins, and for detecting individual cells. Because resonantly coupled light within a photonic crystal can be effectively spatially confined, a photonic crystal surface is capable of supporting large numbers of simultaneous biochemical assays in an array format, where neighboring regions within ˜10 μm of each other can be measured independently. See Li, P., B. Lin, J. Gerstenmaier, and B. T. Cunningham, A new method for label-free imaging of biomolecular interactions. Sensors and Actuators B, 2003.
Given substantial advances in their fabrication and their unique optical properties, photonic crystal-based sensors are under development for a variety of applications. Biosensors are one application. Biosensors incorporating photonic crystal structures are described in the following references, which are hereby incorporated by reference in their entireties: U.S. Pat. Nos. 7,118,710, 7,094,595, and 6,990,259; U.S. Published applications 2007/0009968; 2002/0127565; 2003/0059855; 2007/0009380; 2003/0027327; Cunningham, B. T. J. Qiu, P. Li, J. Pepper and B. Hugh, A Plastic calorimetric Resonant Optical Biosensor for Multi-parallel Detection of Label Free Biochemical Interactions, Sensors and Actuators B, 2002, 85, pgs 219-226.
U.S. Pat. No. 6,707,561 describes a grating-based biosensing technology that is sometimes referred to in the art as Evanescent Resonance (ER) technology. This technology employs a submicron scale grating structure to amplify a fluorescence signal, following a binding event on the grating surface, where one of the bound molecules carries a fluorescent label. ER technology enhances the sensitivity of fluorophore based assays enabling binding detection at analyte concentrations significantly lower than non-amplified assays.
ER technology uses grating generated optical resonance to concentrate laser light on the grating surface where binding has taken place. In practice, a laser scanner sweeps the sensor at some angle of incidence (θ), typically from above the grating, while a detector detects fluoresced light (generally at longer optical wavelength) from the sensor surface. By design, ER grating optical properties result in nearly 100% reflection, also known as resonance, at a specific angle of incidence and laser wavelength (λ). Confinement of the laser light by and within the grating structure amplifies emission from fluorophores bound within range of the evanescent field (typically 1-2 μm). Hence, at resonance, transmitted light intensity drops to near zero.
The spectral width and wavelength of the resonance phenomena describes the important externally measurable parameters of a device. Resonance width refers to the full width at half maximum, in wavelength measure, of a resonance feature plotted as reflectance (or transmittance) versus wavelength. Resonance width also refers to the width, in degrees, of a resonance feature plotted on a curve representing reflectance or transmittance as a function of θ. In practice, one can make adjustments to the incident angle to “tune” the resonance towards maximum laser fluorophore coupling.
In one embodiment of this invention, a biosensor is constructed as a photonic crystal structure which has a periodic surface grating in which a so-called evanescent resonance is created. Conceptually, resonance phenomena can occur in planar dielectric layer gratings where almost 100% switching of optical energy between reflected and transmitted waves occurs when the grooves of the grating have sufficient depth and the radiation incident on the corrugated structure is at a particular angle. This phenomenon is exploited in the sensing area of the platform where that sensing area includes grating structures (e.g., grooves, or holes or posts) of sufficient depth and light is caused to be incident on the sensing area of the platform at an angle such that evanescent resonance occurs in that sensing region. This creates in the sensing region an enhanced evanescent field which is used to excite samples under investigation. It should be noted that the 100% switching referred to above occurs with parallel beam and linearly polarized coherent light and the effect of an enhanced evanescent field can also be achieved with non-polarized light of a non-parallel focused laser beam. Excitation photons incident on the sample (chip, for example) under resonance conditions couple into a thin corrugated surface (such as a metal oxide layer) at the site of incidence. As a result of the transducer geometry, the energy is locally confined into the thin corrugated layer of high refractive index material. Consequently, strong electromagnetic fields are generated at the surface of the chip. The effect has been attributed as evanescent resonance and leads to increased fluorescence intensity of chromophores (fluorescent material) close to the surface of the sensor. The effective field strength can be increased up to 100-fold by the confinement of the available excitation energy, depending on the optical properties of the optical detection system used.
The inventive sensors and method of this disclosure are useful in conjunction with a variety of different types of fluorophores. Such fluorophores have excitation and emission spectra which are typically well characterized and available from the manufacturer, or can be determined experimentally.
Quantum Dots (QDs) are fluorescent, nanometer-sized inorganic semiconductor crystals that have rapidly emerged as an important class of nanomaterials which promise to revolutionize a wide range of nanotechnology-enabled fields. QDs derive their unique optical properties (broad absorption spectrum, narrow, size-tunable emission spectrum, high photostability, quantum efficiency and strong nonlinear response) from quantum confinement effects. These attributes, coupled with the ability to functionalize QDs, has made them important candidates for light sources, solar cells, optical switches and fluorescent probes in sensitive biological assays. The ability to more efficiently excite and extract the light emitted by QDs would thus be of vital importance in realizing high brightness light sources, enhanced nonlinear effects and lowering the detection limits in biological assays.
Fluorescent dyes represent a broad class of organic and inorganic fluorescent molecules that are capable of emitting light. Generally, electrons within the fluorescent molecule are excited from a ground state to an excited state through the absorption of a photon from an external source of illumination. The electron in the excited state may return to the ground state through a variety of mechanisms. One such mechanism is through the release of heat in the form of a phonon. Another such mechanism is through the release of light in the form of a photon. Absorption of energy by the fluorophore occurs at a particular range of incident photon energies (or equivalently wavelengths) that are unique for each type of molecule. Due to conservation of energy, the emitted photon energy must be less than or equal to the energy of the incident photon, and therefore the emitted wavelength must be larger than the incident photon wavelength. Therefore, a fluorescent molecule has two distinct spectra associated with it: the range of wavelengths for which it is capable of absorbing photons, and the range of wavelengths for which it is capable of emitting photons. The difference between the absorption and emission wavelength is known as the Stokes shift.